The present invention relates to a biological assay and a biological assay apparatus.
Biochemical, microbiological, chemical and many other assays are being performed every day in laboratories. While a considerable amount of attention has naturally been placed on such biological cell assaying for humans, this is also be becoming more important in the field of animal welfare and indeed plant production generally.
A rapidly advancing research area in biology is the study of cell receptor-ligand interactions resulting in cell-substratum and cell-cell adhesion followed by subsequent cell migration. The pre-requisite to transendothelial migration of certain cell lines into sites of infection is paramount to the study of inflammatory diseases. This can be briefly summarised as cell flow and rolling, tethering and activation of integrin receptors which is a key recognition step, attachment to the endothelial ligands via activated integrins and finally transendothelial migration or diapedesis. Unfortunately, to date, most of the assay techniques are not particularly successful for the study of these mechanisms. Currently, the majority of studies involving cell rolling and chemokine induced cellular arrest have utilised capillary systems wherein cell flow and shear stress are controlled utilising syringe pumps. Such observations are constrained by a number of factors. Firstly, the relative large ( greater than 100 xcexcm) size of the standard glass capillaries limits the physiological analogies to the proximal microvascular regions. Secondly, such studies can only be utilised to study single end-points and cannot be utilised to examine cell choices in migration Thirdly, optical aberrations related to the spherical geometry of the glass capillary sections limit stage-related in situ (post-fixation) analysis of the intracellular structures (cytoskeleton and signalling molecules). Finally and most importantly, the usual observation periods lie between 5-30 minutes for rolling experiments. Longer studies are required to study subsequent crawling steps on endothelial and extracellular matrix ligands. In this regard, studies relating to the effects of chemokines have largely been limited to cellular arrest on adhesion receptor ligands and have not been extended to the study of cell crawling. For example, specific chemokines have been shown to induce rolling arrest with enhanced binding of lymphocytes to ICAM-1, otherwise known as CD54.
Presently accepted techniques for cell adhesion or binding assays involve the initial coating of a surface of a device with a substrate, typically a protein. Cells are deposited onto the substrate and allowed to settle. Following the settling of the cells, the device is placed on a heating stage at 37xc2x0 C., which is attached to an inverted microscope for visual analysis, or alternatively to a stand-alone heating stage and progression of cell binding can be checked at intervals with the inverted microscope. The duration of these assays may be varied depending on the cell line and choice of substratum. Following cell adhesion, free cells may be washed away and a subsequent cell count may be carried out.
Although these methods provide us with semi-quantitative information regarding a cell type""s affinity for a particular substratum, there is no simple method for quantitative characterisation of binding or methods enabling a prolonged study of cell rolling, the ensuing capture by the substratum and subsequent attachment. Furthermore, direct studies of changes in cell morphology, cell growth and biochemical changes cannot be provided easily with these techniques since, determining the kinetics of attachment and resulting morphological changes requires multiple replicated experiments being analysed at different times.
U.S. Pat. No. 5,998,160 (Berens et al) describes a static assay which unfortunately does not have any consideration of cell flow and rolling.
The ability of T-cells circulating in the bloodstream to adhere to the endothelium, switch to a motile phenotype and penetrate through the endothelial layer is recognised as a necessary requirement for the effective in vivo movement or as it is sometimes referred to, trafficking of specific lymphocyte sub-populations. Motility assays are done in combination with attachment assays since following adhesion; cells are expected to switch to the motile phenotype. Motility assays are assessed by estimating the ratio of cells undergoing cytoskeletal rearrangements and the formation of uropods (extension of the trailing tail). One of the major disadvantages of this and the previous adhesion assays is the geometrical design (microscope slides and multiple well chambers), which does not at all resemble the in vivo situation.
The most commonly used cell transmigration assay is a modified xe2x80x9cBoyden chamberxe2x80x9d assay such as described in U.S. Pat. No. 5,578,492 (Fedun et al). This involves assessing the crossing of a quantity of cells through a microporous membrane under the influence of a chemoattractant, recombinant or cell-derived. Here the diameter of the micropores are less than the diameter of the cells under investigation, such that the cells must deform themselves in order to squeeze through the pores thereby constructing an analogy to the transendothelial migration of cells in physiological circumstances. Once the cells are deposited onto the membrane, the chamber can be incubated for intervals over time at a suitable temperature, usually 37xc2x0. Following this, the bottom chamber or opposite side of the top chamber may be analyzed for cells that have squeezed through the microporous membrane.
U.S. Pat. No. 4,912,057 (Guirguis et al), U.S. Pat. No. 5284753 (Goodwin et al), U.S. Pat. No. 5,302,515 (Goodwin et al), U.S. Pat. No. 5,514,555 (Springer et al) and U.S. Pat. No. 5,601,997 (Tchao) are typical examples of these assays. The main disadvantage of the assays described in those specifications is that the biological process of transmigration through the micropores is difficult to observe due to the geometrical configuration of the apparatus involved. The lens of the optically inverted microscope must be able to focus through the lower chamber and the microporous membrane. This obviously leads to difficulties due to optical aberrations. In effect, the study of the cells morphology changes while transmigrating across the membrane and their subsequent cytoskeletal changes reverting to their former state is a process which is difficult to monitor and record due to limitations with current techniques. In addition, although it is possible to alter such an experiments parameters following the initiation of the experiment, such as the introduction of a second chemoattractant, recombinant or cell-derived, at some specified time after commencing the experiment, it is not possible to distinguish separate effects from each said chemoattractant.
In addition to cell biology studies, the pharmaceutical industry has major problems in the drug screening process and while high throughput screening (HTS) has been extremely successful in the elimination of the large majority of unsuitable drugs, it has not progressed beyond that and usually, after a successful HTS assay, a pharmaceutical company may still have 7,000 possible drugs requiring assessment. This requires animal trials and anything that can be done to reduce the amount of animal trials is to be desired. Thus, there is a need for new techniques for drug testing in the pharmaceutical industry. The current proposals are to screen the physiological response of cells to biologically active compounds such as described in U.S. Pat. No. 6,103,479 (Taylor). This again, unfortunately, is still a static test. Since the cells are spatially confined with the drug, there may be a reaction but it may not necessarily take place when the cells are free to flow relative to the drug as in, for example, the microcapillaries of the body. There are other disadvantages such as the transport and subsequent reaction of the drug following its injection into the animal. Probably the most important disadvantage is that it does not in any way test, in a real situation, drug efficacy.
Finally, there are no techniques at the present moment for performing assays to test the interaction of a large number of chosen compounds with living cells while the cells or compounds mimic the in vivo situation of continuous flow.
While in the description herein, the examples all refer to animal cells and indeed mainly human cells, the invention equally applies to plan cells. The term xe2x80x9csample liquidxe2x80x9d refers to a suspension of living cells within a suitable carrier liquid which is effectively a culture medium. More than one cell type may be in suspension. Further, the term xe2x80x9creagent liquidxe2x80x9d could be any liquid from a drug under assessment, a poison, a cell nutrient, chemoattractant, a liquid containing other cells in suspension or indeed any liquid who""s effect the sample liquid requires assessment.
The present invention is directed towards providing such methods and apparatus for performing such assays.
The present invention provides a biological assay method comprising:
delivering a sample liquid of a suspension of cells at a controlled steady flow rate through a biochip in the form of an elongate enclosed microchannel;
causing an externally generated test to be carried out on the sample liquid as it is being delivered through the biochip; and
examining the sample liquid over time to observe the effect of the test on the sample.
The externally generated test can be carried out in many ways, for example, it can comprise coating the internal bore of the biochip with a protein which could, for example, be an extracellular matrix ligand or could be formed by an endothelium layer which in turn would be formed by seeding the biochip with endothelial cells allowing the cells to grow on the walls. The cells can be taken from an animal or indeed most often from a human, but could also be from a plant. The bore of the biochip, in certain tests, is substantially the same size as the post capillary venules of an animal or, more particularly, a human. With such a method, for example, one can have tests for cell flow, rolling, tethering and migration of previously adhered cells, and adhesion. All of these may be recorded in any suitable manner. It is envisaged that the velocity of the delivery of the sample liquid may be varied to provide different test conditions or the velocity of the delivery of the sample liquid can be increased until previously adhered tests are removed and then the velocity forms a measure of the adherence. Alternatively, a separate flushing liquid may be introduced to remove previously adhered cells, the velocity of the flushing liquid forming a measure of the adherence. Needless to say, after cells have been adhered to the protein, the sample liquid could be replaced by a reagent liquid and the effect of the reagent liquid could be observed. The reagent liquid could be any suitable liquid. One could be, for example, an adhesion detachment reagent liquid and thus the effect of this on the previously adhered cells could be monitored. Needless to say, any reagent liquid may be delivered simultaneously with the sample liquid through the biochip to achieve various tests. For example, it would be possible to deliver a reagent liquid at a controlled steady flow rate through another microchannel connected to the first microchannel, the channels being connected intermediate their ends by an interconnecting channel. The fluid pressure of the liquids could be chosen so as to cause a diffusion of the reagent through the interconnecting channel or alternatively the fluid pressures could be maintained equal to prevent diffusion of the reagent. Similarly, the channels may be connected intermediate their ends by an interconnecting channel having a restricted entry throat, which restricted entry throat would preferably have a cross sectional area less than that of a cell when the cell is freely suspended in the sample liquid. This would be a very good way of studying the mechanisms involved in cell migration from the endothelium to the extracellular matrix.
In other embodiments, the bore of the microchannel could be provided with a hydrophobic coating such as liquid silicon.
It is envisaged that more than one cell type may be held in suspension as this often happens in practice and indeed in many instances, it may be advantageous to deliver a reagent liquid and a sample liquid through the microchannel to form multilaminar flow and then if the reagent liquid comprises a chemoattractant suitable for one of the types of the cell, it will be possible to effectively separate that particular type of cell from the sample.
Further, the invention envisages a method in which the biochip comprises two microchannels, one a feeding microchannel having a cell reservoir intermediate its ends and the other a reactant microchannel connected to the reservoir by a connecting means comprising:
storing cells in the cell reservoir;
feeding and growing the cells in the cell reservoir by delivering a culture medium through the feeding microchannel; and
delivering reagent liquid through the reactant microchannel.
The reagent liquid could, for example, be one or more of a chemoattractant, toxic substance or pharmaceutical preparation and these could be recombinant or cell derived.
It is envisaged that a plurality of tests can be carried out simultaneously using the one sample liquid forming portion of a large sample and using different test conditions or alternatively, a plurality of the same tests may be carried out using different sample liquids and the same test conditions.
According to the invention, there is provided a biochip comprising:
an elongate main microchannel;
an inlet port mounted on the proximal end of the main microchannel;
an outlet port adjacent its distal end;
a separate liquid feeder microchannel connected to the main microchannel, the feeder microchannel having an inlet port; and
an outlet feeder port connecting the feeder microchannel and the main microchannel.
Ideally, the outlet port between the feeder microchannel and the main microchannel has a restricted throat. Further, there can be produced a biochip comprising two separate main microchannels and a connecting microchannel connecting the two separate main microchannels. Such separate microchannels can be parallel, diverge towards each other and indeed the connecting channel may also have a restricted throat or the channel itself may just have a restricted cross section.
Further, there is provided a biochip comprising:
two separate main microchannels; and
a common microchannel connected to the two main microchannels to provide an extension of the two main microchannels.
This common microchannel can feed two further microchannels and indeed the microchannel can comprise a main microchannel and a take-off microchannel intermediate its ends, the take-off microchannel having an entrance which projects into the main microchannel to divert flow from the main microchannel into the take-off microchannel. Further, a microwell can be incorporated in a microchannel forming part of a biochip, which microwell may have connected to it a further feeder microchannel delivering into and out of the microwell, the feeder microchannel having an inlet port adjacent its proximal end and an outlet port adjacent is distal end.
It is envisaged that the microchannel according to the present invention will generally have a planar top wall to allow good optical properties for examination under a microscope and generally speaking, the microchannel comprises planar top, bottom and side walls which side walls taper outwards and upwards away from each other. Ideally, the top wall is removable and is formed from a plastics film.
Preferably, each port has a bubble release port and valve associated therewith. The cross sectional area of the microchannel is between 25 xcexcm2 to 10,000 xcexcm2 and preferably greater than 400 xcexcm2.
It is envisaged that assemblies comprising a plurality of biochips as described above will be formed on the one base sheet and will preferably have various common feeder microchannels having ports therein. The advantage of a whole lot of biochips all on the one sheet is that they can be readily easily examined by the one microscope.